Method for reducing hypertension and heart failure

ABSTRACT

A method is provided for reducing hypertension and/or heart failure in a mammal. Preferably, the method is used to treat or prevent tissue damage to a human heart. The method includes administering an effective amount of a Jak2 inhibitor, preferably a tyrphostin, such as AG490.

[0001] This application is a continuation-in-part of U.S. Ser. No.09/945,192 filed on Aug. 31, 2001, the entire specification is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to methods for reducing hypertension,hypertrophy, ischemia, and/or heart failure.

[0003] Cardiovascular disease is the leading cause of death in theWestern world, resulting in an estimated annual death toll of more thanten million people. Such diseases, such as chronic hypertension (highblood pressure), left ventricular hypertrophy (enlargement of theheart), and myocardial ischemia (cardiac cell injury) can culminate inheart failure.

[0004] The most prevalent cardiovascular disorder that contributes toheart failure is hypertension, which is a disease largely of thevasculature. The complex pathogenesis of hypertension is not fullyunderstood, although it is believed that functional and/or structuralchanges in the blood vessels are the cause.

[0005] High blood pressure is a significant health problem for severalreasons. First, only one-third of the patients receiving treatment havetheir illness under control. Furthermore, one-third of the population inthe United States are estimated to have undetected hypertension (Kaplan,(1998) Clinical Hypertension. Baltimore: Williams & Williams).

[0006] The consequences of hypertension (e.g., hypertrophy, heartfailure, coronary heart disease, aortic disease, and renal failure,etc.) are widespread and can be devastating. Victims can remainasymptomatic until much damage has already occurred. Furthermore, thedetrimental effects of blood pressure increase continuously as thepressure increases.

[0007] As stated above, one consequence of hypertension is generallyhypertrophy. Cardiac hypertrophy is an increase in the size of theheart. In humans, hypertrophy, is the compensatory response of themyocardium (cardiac muscle) to increased work as a result of an increasein blood pressure or blood volume (hemodynamic overload). The myocardiumcan increase in size but is not capable of increasing cell number.

[0008] Two patterns of hypertrophy can occur depending on the stimulus,either pressure-overloaded hypertrophy or volume-overloaded hypertrophy.Pressure-overloaded hypertrophy typically occurs as a result ofhypertension. The ventricles develop concentric hypertrophy, and exhibitan increased ratio of wall thickness to cavity radius.

[0009] Volume-overloaded hypertrophy generally occurs as a result of adefect in one of the valves of the heart. The ventricles develophypertrophy with dilatation (eccentric hypertrophy), resulting in aproportionate increase in ventricular radius and wall thickness.

[0010] Initially, the development of cardiac hypertrophy is advantageoussince it results in the addition of sarcomeres (contractile units),thereby reducing ventricular wall stress to normal levels (Ruwhof etal., (2000) Cardio. Res., 47:23-37). The increase in the number ofsarcomeres leads to augmentation in the overall weight and size of theheart.

[0011] With prolonged hemodynamic overload, however, when thehypertrophied heart can no longer meet the increased demand in workload,the heart begins to dilate, stretching the sarcomeres and increasing theforce of contraction and stroke volume. The increased stretching of themyocytes further perpetuates the hypertrophy.

[0012] Hypertrophy of the myocardium may become increasingly harmful dueto the increased metabolic requirements of the enlarged heart. Molecularchanges have been observed in the myocytes during development ofmyocardial hypertrophy. Such changes include the rapid induction ofproto-oncogenes and heat shock protein genes, quantitative andqualitative changes in gene expression, and increased rate of proteinsynthesis (Ruwhof et al., (2000) Cardio. Res., 47:23-37). Changes thatoccur in the hypertrophied heart may contribute to the development ofheart failure. Moreover, ischemic heart disease and arrhythmias maydevelop, increasing the risk of death.

[0013] A different type of heart disease occurs as a result of ischemia.Ischemia is an imbalance between the supply and demand of the heart foroxygenated blood. In addition to insufficient oxygen, ischemia is alsocaused by a reduced availability of nutrient substrates and inadequateremoval of metabolites. In the majority of cases, myocardial ischemiaoccurs as a result of the narrowing or obstruction of an artery due toatherosclerosis. Four ischemic syndromes may result depending on therate of development and severity of the arterial narrowing and themyocardial response. The ischemic syndromes are angina pectoris,myocardial infarction, chronic ischemic heart disease, and suddencardiac death.

[0014] The cardiac diseases described above can ultimately impaircardiac function and result in heart failure. Development of heartfailure usually occurs slowly, often over many years. The heartgradually loses its ability to pump blood and therefore works lessefficiently. As such, heart failure is typically defined as a clinicalsyndrome in which the heart is unable to maintain an output sufficientfor the metabolic requirements of the tissues and organs of the body.

[0015] The tissue and systemic renin-angiotensin systems play a majorrole in regulation of pathological cardiovascular functions, such as inhypertension (Raizada et al., (1993) Cellular and Molecular Biology ofthe Renin-Angiotensin System, 515-555), left ventricular hypertrophy(Lavie et al., (1991) Drugs 42:945-946), ischemic dilatedcardiomyopathy, and heart failure (Raynolds et al., (1993) Lancet342:1073-1075). The renin-angiotensin system also exists in other organsand tissues, including the heart, kidneys, prostate, brain, intestines,and the vasculature.

[0016] Normal homeostatic levels of a number of hemodynamic properties,such as blood pressure, blood volume, and vascular tone, are maintainedby the renin-angiotensin system. Renin is an enzyme that was firstisolated from the kidneys over a hundred years ago. Angiotensinogen iscleaved by renin to yield the inactive decapeptide angiotensin I. Anenzyme is present in the vascular endothelium, especially in the lungs.The enzyme is angiotensin converting enzyme (ACE), which cleaves off twoamino acids from angiotensin I to form the octapeptide, angiotensin II.

[0017] Angiotensin II is prominently involved in virtually all aspectsof the renin-angiotensin activity. The angiotensin II then exerts itseffects on target organs and tissues by binding its transmembrane domainG-protein coupled receptor (AT₁ and/or AT₂).

[0018] Binding of angiotensin II to its receptor can activate severaldifferent intracellular signal transduction pathways that use thewell-known signal transducers, such as protein kinase A, protein kinaseC, MAP kinase, and src (Sadoshima et al., (1993) Circ. Res. 73:413-423;Duff et al., (1995) Cardiovasc. Res. 30:511-517; Booz et al., (1995)Cardiovasc. Res. 30:537-543; Schieffer et al., (1996) Hypertension27:476-480; Bernstein et al., (1996) Trends Cardiovasc. Med. 6:179-197).

[0019] In addition to these signal transduction pathways, angiotensin IIalso activates the Janus-associated kinase/signal transducer andactivator of transcription (Jak/STAT) pathway. The components of theJak/STAT pathway are present in a latent state in the cytoplasm ofunstimulated cells. Binding of angiotensin II to its receptor leads toactivation of Jak, a tyrosine kinase that phosphorylates STAT proteinsand allows them to translocate to the nucleus. Within the nucleus, thephosphorylated STAT functions as a transcription factor (Ihle (1996)Cell 84:331-334) that recognizes and binds, in a sequence-specificfashion, to cis-regulatory elements in the promoter of target genes.

[0020] In mammals, the Jak family consists of Jak1, Jak2, Jak3, andTyk2. Seven STAT proteins have been identified in mammalin cells, STAT1,STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6.

[0021] Jaks are crucial components of diverse signal transductionpathways that govern important cellular functions, including cellsurvival, proliferation, differentiation and apoptosis. Interfering withJak activity may lead to the loss of a vital signal transductionpathway, thereby disrupting normal cellular processes needed for cellsurvival. Therefore, it is important to selectively inhibit particularJaks that are involved in various disease states. For example, Jak2 hasbeen suggested to be involved in the upregulation of angiotensinogenpromoter activity in hypertrophy and ischemia (Mascareno E, et al.(2000) Mol. Cell. Biochem. 212:171; and Mascareno E, et al (2001)Circulation 104:1).

[0022] Inhibitors of Jaks include tyrphostins, which are a class ofcompounds that inhibit protein tyrosine kinases. The tyrosine kinasesthat are inhibited depends on the substituents that are present on thetyrphostin.

[0023] One particular tyrphostin, AG490, selectively inhibits Jak2 andhas been proposed for treating cancer (Meydan N, et al. (1996) Nature379:645). Administration of tyrphostin AG490 has been suggested toafford cardioprotection to hearts subjected to ischemia/reperfusion(Mascareno E, et al. (2000) Mol. Cell. Biochem. 212:171 and Mascareno E,et al (2001) Circulation 104:1). However, the reference does notdisclose treating hypertension and/or heart failure with tyrphostinAG490.

[0024] Tyrphostin AG556 is a protein tyrosine kinase inhibitor thatreduces myocardial damage due to ischemia (Altavilla D., et al, (2000)Life Sciences 67:2615). There is no indication that tyrphostin AG556 isa selective Jak2 inhibitor. The lack of selectively is a problem sinceit can lead to side effects.

[0025] There has been an ongoing search for effective long-termtreatments for myocardial dysfunction. Currently, treatments includeadministering drugs, such as vasiodilators, beta-blockers, free-radicalscavengers, and calcium antagonists. Another type of treatment issurgery and includes by-pass surgery and angioplasty. Virtually all ofthese methods have been ineffective for favorable long-term results.

[0026] Heart muscle cannot currently be regenerated. As a consequence,affected individuals must contend with damaged heart tissue for the restof their lives. Therefore, restoring normal cardiac function to heartmuscles damaged by cardiovascular disease has been a long-term goal ofcardiology.

[0027] Therefore, there is an immediate need for therapeutic agents thatprevent and/or reverse the damage caused by myocardial dysfunctionwithout harming healthy cells.

SUMMARY OF THE INVENTION

[0028] These and other objectives have been met by providing a methodfor reducing hypertension in a mammal at risk for said hypertension. Themethod comprises administering to said mammal an effective amount of aselective Jak2 inhibitor.

[0029] In another embodiment, the invention provides a method forreducing hypertrophy of an organ in a mammal at risk for saidhypertrophy. The method comprises administering to said mammal aneffective amount of a pharmaceutical composition comprising a selectiveJak2 inhibitor.

[0030] In yet another embodiment, the invention relates to a method forreducing ischemia of an organ in a mammal at risk for said ischemia. Themethod comprises administering to said mammal an effective amount of apharmaceutical composition comprising a selective Jak2 inhibitor.

[0031] In a further embodiment, the invention relates to a method forreducing heart failure in a mammal at risk for said heart failure. Themethod comprises administering to said mammal an effective amount of aselective Jak2 inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1: depicts a model for transverse aortic constriction (TAC)in mice.

[0033]FIG. 2: depicts cardioprotection of left ventricular hypertrophy(LVH) by tyrphostin AG490. A) Visual inspection of cross section of theheart demonstrates a decrease in LVH in tyrphostin AG490 treatedanimals. B) Histogram demonstrates a decrease in ratio of heart weightto body weight in tyrphostin AG490 treated animals. C) Light microscopyof cardiomyocytes of left ventricle demonstrates a decrease inhypertrophy in tyrphostin AG490 treated animals.

[0034]FIG. 3: depicts ANF inhibition by tyrphostin AG490 during cardiachypertrophy.

[0035]FIG. 4A: depicts the effects of tyrphostin AG490 on myocardialfunction.

[0036]FIG. 4B: depicts reduction of infarct size by tyrphostin AG490during ischemia/reperfusion.

[0037]FIG. 4C: depicts reduction of apoptosis of cardiomyocytes bytyrphostin AG490 during ischemia/reperfusion.

[0038]FIG. 5: depicts up-regulation of angiotensinogen mRNA duringischemia/reperfusion (I/R) is mediated by STATs. A) Angiotensinogen mRNAis increased during ischemia/reperfusion. B) St-domain/STAT bindingactivity is increased in hearts subjected to ischemia/reperfusion. C)STAT5A and STAT6 are activated in ischemic hearts.

[0039]FIG. 6A: depicts Jak2 inhibition by tyrophostin AG490 duringischemia/reperfusion (I/R).

[0040]FIG. 6B: depicts reduction of St-domain/STAT binding activity bytyrphostin AG490 in hearts subjected to ischemia/reperfusion.

[0041]FIG. 6C: depicts inhibition of angiotensinogen mRNA by tyrphostinAG490 in hearts subjected to ischemia/reperfusion.

[0042]FIG. 7: depicts Jak2 as a potent activator of angiotensinogen geneexpression.

[0043]FIG. 8: depicts attenuation of angiotensinogen mRNA expression invivo by tyrphostin AG490.

[0044]FIG. 9: depicts attenuation of hypertension by tyrphostin AG490 inspontaneously hypertensive rats.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention is based on the discovery by the inventorsthat a specific signaling pathway is responsible for the onset andmaintenance of the renin-angiotensin system in hypertension,hypertrophy, and ischemia. The inventors have discovered that theactivation of Jak2, during hypertension, hypertrophy and ischemiaactivates specific STAT proteins, specifically STAT 3, STAT5A and STAT6.Moreover, the inventors have discovered that administration of a Jak2inhibitor significantly reduces hypertension, and myocardial damagecaused by hypertrophy and ischemia Administration of a Jak2 inhibitorcan also significantly reduce heart failure.

[0046] Hypertension

[0047] In one embodiment, the invention provides a method for reducinghypertension in a mammal at risk for hypertension. The method comprisesadministering to said mammal an effective amount of a selective Jak2inhibitor.

[0048] Hypertension is typically a disease of the vasculature thatcauses high blood pressure. High blood pressure usually occurs as aresult of resistance in the blood vessels to blood flow. The resistancemay, for example, be due to functional or structural changes to theblood vessels (e.g., artherosclerosis, arteriolosclerosis, andarteriolitis). The greater the resistance to blood flow, the harder theheart must work to maintain an adequate blood flow to the body, thusresulting in a higher blood pressure.

[0049] Hypertension can be defined by an elevated diastolic and/orsystolic blood pressure. The healthy (e.g., normal) and elevateddiastolic and/or systolic blood pressure for a particular mammal isknown to those skilled in the art.

[0050] For example, in humans, high blood pressure is typically definedwhen the sustained diastolic pressure is greater than about 85 mm Hg, inmore serious cases greater than about 100 mm Hg, and in the most seriouscases greater than about 115 mm Hg. Using systolic pressure as ameasurement, in humans, high blood pressure is typically defined whenthe sustained systolic pressure is greater than about 140 mm Hg, in moreserious cases greater than about 150 mm Hg, and in the most seriouscases greater than about 160 mm Hg.

[0051] Hypertension may be assessed by any method known to those skilledin the art. For example, blood pressure may be measured with a bloodpressure cuff and gauge or with a blood pressure monitor (e.g.,electronic sphygmomanometer).

[0052] Hypertrophy

[0053] In another embodiment, the invention relates to a method forreducing hypertrophy of an organ in a mammal at risk for hypertrophy.The method comprises administering to said mammal an effective amount ofa pharmaceutical composition comprising a selective Jak2 inhibitor.

[0054] Hypertrophy is the enlarging of an organ. The increase in sizemay, for example, be due to an increase in workload due to some physicaldefect in the organ itself or one of the biological systems supportingthe organ.

[0055] Several organs are subject to hypertrophy. Some examples includethe heart, kidney, and prostate.

[0056] Myocardial hypertrophy, for example, is hypertrophy of the heart,which is typically caused by either myocardial valve damage or highblood pressure. Myocardial hypertrophy may also result from a dilationor expansion of the heart in response to heart muscle damage that causesweak muscle action. Hypertrophic damage may lead, for example, tomyocardial infarction, congestive heart failure, and cardiomyopathy.

[0057] Left ventricular hypertrophy (LVH) is the medical term forenlargement of the left ventricle of the heart. The left ventricle isthe heart's main pumping chamber, and pumps oxygenated blood via theaorta through the systemic circulation.

[0058] Hypertrophy may be assessed, for example, by any method known tothose skilled in the art. For example, the weight of the organ relativeto the body weight of the mammal may be expressed as a ratio, asdescribed in Example 1 and depicted in FIG. 2B.

[0059] Ischemia

[0060] In a further embodiment, the invention relates to a method forreducing ischemia of an organ in a mammal at risk for ischemia. Themethod comprises administering to said mammal an effective amount of apharmaceutical composition comprising a selective Jak2 inhibitor.

[0061] Ischemia is a deficiency of oxygenated blood. The deficiency ofblood may, for example, be caused by functional constriction orobstruction of a blood vessel. The lack of oxygen and/or reducedavailability of nutrient substrates and inadequate removal ofmetabolites may result in tissue damage, for example, apoptosis and/ornecrosis of cells.

[0062] Several organs are subject to ischemia. Some examples include,but are not limited to, the heart, brain, kidney, and intestines.

[0063] Ischemic heart disease is often caused by a reduction in coronaryblood flow relative to myocardial demand. The reduction in blood flowmay result from a variety of reasons, and typically occurs as a resultof atherosclerosis.

[0064] As a result of ischemic damage to the heart muscle, the damagedarea ceases to contract. Symptoms of such damage include, but are notlimited to, cardiac arrhythmias, angina, myocardial infarction,congestive heart failure, and sudden cardiac death.

[0065] Ischemia may be assessed by any method known to those skilled inthe art. An assessment of ischemic damage may be made, for example, bymeasuring the infarct (scar) size of the organ, as described in Example2 and depicted in FIG. 4B.

[0066] Heart Failure

[0067] In yet another embodiment, the invention relates to a method forreducing heart failure in a mammal at risk for heart failure. The methodcomprises administering to said mammal an effective amount of aselective Jak2 inhibitor.

[0068] Heart failure is a clinical syndrome resulting from disturbancesin cardiac output or from increased venous pressure. The disturbances incardiac output or increased venous pressure can be due to dilatedcardiomyopathy, myocardial fibrosis, deposition of amyloid, constrictivepericarditis, hypertension, hypertrophy and/or ischemia.

[0069] Injury to the heart muscle by hypertension, hypertrophy and/orischemia often decreases the ability of the heart to contract.Therefore, the heart cannot pump with ample force to push a sufficientamount of blood into the circulation.

[0070] In addition, injury to the heart muscle can prevent the heartfrom fully relaxing. As a consequence, the heart cannot properly fillwith blood.

[0071] Heart failure may be assessed by any method known to thoseskilled in the art. An assessment of heart failure may be made, forexample, by the symptoms associated with heart failure, such as chestpain, shortness of breath, excess fluid in the lungs, fatigue, and/orswollen ankles and feet. Instruments can be used to quantitate thesymptoms of heart failure and are known to those skilled in the art. Forexample, a caliper can be used to measure the amount of swelling in theankles and feet and a spirometer can be used to measure lung capacity todetermine shortness of breath.

[0072] An assessment of heart failure can also be made by measuringheart function. Instruments used to evaluate heart function include anelectrocardiogram (i.e., measures electrical activity of a heartbeat) orechocardiography (measures abnormal heart size, shape, movement and/oramount of blood pumped out of the heart when the heart contracts).

[0073] Effects of Treatment

[0074] The methods of the invention result in inhibition of Jak2,thereby reducing hypertension, hypertrophy, ischemia, and/or heartfailure.

[0075] Reducing hypertension means a significant reduction in theelevated blood pressure of a mammal relative to a healthy bloodpressure. Hypertension is considered significantly reduced if theelevated diastolic and/or systolic pressure is reduced by at least about10%, preferably at least about 25%, more preferably at least about 50%,even more preferably at least about 75%, and most preferably about 100%(e.g., bringing the pressure down to normal levels).

[0076] Reducing hypertrophy of an organ means a significant reduction inthe size of a hypertrophic organ relative to a healthy organ. Reducingischemia of an organ means a significant reduction in the infarct sizeof an ischemic organ. Hypertrophy or ischemia is consideredsignificantly reduced if the size of the hypertrophic organ or theinfarct size of the ischemic organ is reduced by at least about 10%,preferably at least about 25%, more preferably at least about 50%, evenmore preferably at least about 75%, and most preferably about 100%.

[0077] Reducing heart failure means a significant reduction in thesymptoms of heart failure or heart function relative to a healthy heart.Heart failure is considered significantly reduced if the symptoms ofheart failure or heart function is reduced by at least 10%, preferablyat least about 25%, more preferably at least about 50%, even morepreferably at least about 75%, and most preferably about 100%.

[0078] Any manmmal may be treated in accordance with the invention.Mammals include, for example, humans, baboons and other primates, aswell as pet animals such as dogs and cats, laboratory animals such asrats and mice, and farm animals such as horses, sheep, and cows.

[0079] A mammal at risk for hypertension, hypertrophy, ischemia, and/orheart failure may be susceptible for any number or reasons, including agenetic predisposition and/or environmental insult. Some examples ofreasons for susceptibility to hypertension include, but are not limitedto, familial history of high blood pressure, atherosclerosis,arteriolosclerosis, arteriolitis, diet and lifestyle, and side effectsof medication. Some examples of reasons for susceptibility tohypertrophy include, but are not limited to, familial history of highblood pressure, valvular heart disease, and side effects of medication.Valvular heart disease includes, for example, congenital heart diseaseand rheumatic heart disease. Some examples of reasons for susceptibilityto ischemia include, but are not limited to, familial history ofatherosclerosis, diet and lifestyle, surgical procedures, and sideeffects of medication. Some example of reasons for susceptibility toheart failure include, but are not limited to, dilated cardiomyopathy,myocardial fibrosis, deposition of amyloid, constrictive pericarditis,hypertension, hypertrophy and ischemia.

[0080] Jak2 Inhibitors

[0081] A Jak2 inhibitor is any compound that selectively inhibits thephosphorylation of the Jak2 protein in the Jak/STAT pathway. Thecompound may directly inhibit Jak2, or a component upstream of Jak2. Theinhibition of the Jak2 protein must be sufficient to substantiallyinhibit and preferably prevent the Jak/STAT cascade.

[0082] The Jak2 inhibitor may be any type of compound. For example, thecompound may be a small organic molecule or a biological compound, suchas an antibody or an enzyme.

[0083] Examples of Jak2 inhibitors include some members of a class ofsmall organic molecules called tyrphostins. Tyrphostins inhibit theactivity of protein tyrosine kinases and have the basic structure shownin structure 1 below:

[0084] More than one hundred tyrphostins have been synthesized.

[0085] The tyrphostin may be any tyrphostin that selectively inhibitsJak2. Some examples of tyrphostins include the various structuresdescribed in Meydan et al., (1996) Nature, 379:645-648; Levitzki et al,(1995) Science, 267:1782-1788; and PCT application WO 98/06391. Thesestructures are incorporated herein by reference.

[0086] A preferred class of tyrphostins for use are those compoundsrepresented by structure 1 wherein:

[0087] R₁═C₆H₅—CH₂—NH;

[0088] R₂ and R₃═H, OH, lower alkyl, F, NO₂, CF₃, C₆H₅—SO₂, O—R₄,O—CO—R₄, or R₄

[0089] R₄=phenyl or lower alkyl; and

[0090] lower alkyl=C₁-C₄ branched or unbranched alkyl (for example,methyl or ethyl).

[0091] R₂ and R₃ may be the same or different except R₂ and R₃ cannotboth be H. Preferably, R₂ and R₃ are OH. The preferred compound hasR₁═C₆H₅—CH₂—NH, R₂═OH, and R₃═OH. The preferred compound is known asTyrphostin AG490, which is a selective, specific, and potent Jak2protein tyrosine kinase inhibitor. The structure of AG490 is shown asstructure 2 below:

[0092] The tyrphostins may be made by methods known in the art, forexample, as described in the PCT application WO 98/06391. Briefly, thetyphostins may be synthesized by Knoevenagel condensation of theappropriate benzaldehyde with malononitrile, the appropriate substitutedamide, or other appropriate Knoevenagel condensation partner.

[0093] A compound is considered a selective inhibitor of Jak2 when thecompound inhibits Jak2 activity to an extent significantly greater thanit inhibits the activity of other members of the Jak family, e.g., Jak1,Jak3, and Tyk2. Preferably, the selective inhibitor inhibits Jak2 atleast 2-fold more than it inhibits other members of the Jak family, morepreferably at least about 5-fold more, and most preferably at leastabout 10-fold more.

[0094] Methods for screening for compounds that inhibit members of theJak family are known in the art. For example, a phosphotyrosine assay isdescribed in Example 5 and depicted in FIG. 6A. See also MolecularCloning A Laboratory Manual by J. Sambrook and D. W. Russel, 2001.

[0095] Jak2 inhibitors as defined herein also include pharmaceuticallyacceptable salts. As used herein, pharmaceutically acceptable salts maybe formed by treating the compounds identified above with salt-formingacids and bases which do not substantially increase the toxicity of thecompound.

[0096] Compositions

[0097] In a preferred embodiment, the Jak2 inhibitor is administered ina pharmaceutical composition. The pharmaceutical composition may bemanufactured by known means. The pharmaceutical compositions arepreferably sterile, non-pyrogenic and isotonic preparations, optionallywith one or more of the pharmaceutically acceptable additives listedbelow.

[0098] The pharmaceutical composition may be any composition suitablefor pharmaceutical use in a mammal, especially a human. The compositionmay, for example, be in the form of a solid, a solution, or asuspension.

[0099] Pharmaceutical compositions of the Jak2 inhibitors of theinvention are preferably stable compositions which may comprise one ormore of the following: a stabilizer, a surfactant, preferably a nonionicsurfactant, and optionally a salt and/or a buffering agent. Thepharmaceutical composition may be in the form of an aqueous solution, orin a lyophilized form.

[0100] The stabilizer may, for example, be an amino acid, such as forinstance, glycine; or an oligosaccharide, such as for example, sucrose,tetralose, lactose or a dextran. Alternatively, the stabilizer may be asugar alcohol, such as for instance, mannitol; or a combination thereof.Preferably the stabilizer or combination of stabilizers constitutes fromabout 0.1% to about 10% by weight of the Jak2 inhibitor.

[0101] The surfactant is preferably a nonionic surfactant, such as apolysorbate. Some examples of suitable surfactants include Tween20,Tween80; a polyethylene glycol or a polyoxyethylene polyoxypropyleneglycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10%(w/v).

[0102] The salt or buffering agent may be any salt or buffering agent,such as for example, sodium chloride, or sodium/potassium phosphate,respectively. Preferably, the buffering agent maintains the pH of thepharmaceutical composition in the range of about 5.5 to about 7.5. Thesalt and/or buffering agent is also useful to maintain the osmolarity ata level suitable for administration to a human or an animal. Preferably,the salt or buffering agent is present at a roughly isotonicconcentration of about 150 mM to about 300 mM.

[0103] The pharmaceutical compositions of the present invention mayadditionally contain one or more conventional additive. Some examples ofsuch additives include a solubilizer such as for example, glycerol; anantioxidant such as, for example, benzalkonium chloride (a mixture ofquaternary ammonium compounds, known as “quats”), benzyl alcohol,chloretone or chlorobutanol; anaesthetic agent such as for example amorphine derivative; or an isotonic agent etc., such as described above.As a further precaution against oxidation or other spoilage, thepharmaceutical compositions may be stored under nitrogen gas in vialssealed with impermeable stoppers.

[0104] An effective amount of a Jak2 inhibitor is the amount whichreduces hypertension, hypertrophy, ischemia, and/or heart failure.Optimal doses can be determined by those skilled in the art based on anumber of parameters including, for example, age, sex, weight, severityof the condition being treated, the compound being administered, and theroute of administration. For example, an effective amount of Jak2inhibitor can be that amount that would produce a blood serumconcentration (volume level) of between about 0.01 μM to about 50 μM,preferably between about 0.05 to 10 μM, and more preferably betweenabout 1.0 μM to about 5 μM.

[0105] Administration

[0106] The Jak2 inhibitor can be administered by any suitable method, asis known in the art. For example, the Jak2 inhibitor can be administeredtopically or systemically. Systemic administration is preferred.Administration using controlled release delivery systems, as is known inthe art, is also contemplated herein.

[0107] Systemic administration includes both parenteral and enteralroutes. For example, Jak2 inhibitors such as tyrphostins can easily beadministered intravenously, which is a preferred route of delivery.Intravenous administration can be accomplished by mixing the Jak2inhibitor in a suitable pharmaceutical carrier (vehicle) or excipient asunderstood by practitioners in the art.

[0108] Oral or enteral administration includes, for example,formulations such as tablets, capsules, pills, troches, elixirs,suspensions, syrups, wafers, chewing gum and the like.

[0109] The Jak2 inhibitor may be administered as a protective agentbefore hypertension, hypertrophy, ischemia, and/or heart failure occurs.For example, the Jak2 inhibitor may be used as a prophylactic treatmentto prevent hypertension, hypertrophy, ischemia, and/or heart failure ina mammal at risk for hypertension, hypertrophy, ischemia, and/or heartfailure. To prevent heart failure, for example, the Jak2 inhibitor maybe administered to a mammal suffering from hypertension.

[0110] In another embodiment, the Jak2 inhibitor may be administeredafter the hypertension, hypertrophy, ischemia, and/or heart failureoccurs in order to minimize and/or reverse, as well as to prevent,further damage resulting from hypertension, hypertrophy, ischemia,and/or heart failure. When administering the Jak2 inhibitor afterhypertension, hypertrophy, ischemia, and/or heart failure has occurred,it is preferred that the Jak2 inhibitor be administered as soonthereafter as possible. It is particularly preferred to administer aJak2 inhibitor before hypertension occurs to prevent any damage. Jak2can also be administered while the hypertension, hypertrophy, ischemia,and/or heart failure is occurring.

[0111] Without being bound by theory, it is believed that the methods ofthe invention described can inhibit the activation of Jak2 and thereforeinterfere with the maintenance of the autocrine loop of therenin-angiotensin system, thereby acting as a protective agent.

EXAMPLE 1

[0112] This example demonstrates cardioprotection from left ventricularhypertrophy by Tyrphostin AG 490.

[0113] Pressure overload was produced by transverse aortic constriction(TAC) to induce left ventricular hypertrophy (FIG. 1). Briefly, maleC57/BL6 mice, weighing 20 to 24 grams, were anesthetized byintra-peritoneal injection of a cocktail of ketamine (100 mg/kg) andxylazine (5 mg/ml). The mice were shaved, restrained, and orallyintubated (under direct vision via a vertical cervical incision) using a22 guage blunt feeding needle. Respiration was artificially controlled(tidal volume of 0.1 to 0.3 ml) at a respiratory rate of 110 to 150breaths/minute using a ventilator (Harvard Apparatus Rodent Ventilator,model 683). A median sternotomy was performed and the sternum retracted.The thymus was retracted anteriorly and the aortic arch identified andligated (using 8.0 nylon suture; Ethicon) between the innominate andleft common carotid artery with an overlying 27-guage needle; and thenthe needle removed to leave a discrete region of stenosis. The chest wasthen closed in two layers (using 6.0 vicryl suture, Ethicon) and thepneumothorax evacuated. Some mice were subjected to a sham operation inwhich the aortic arch was visualized but not banded. The mice were thenextubated and monitored post-op for 3 to 12 hours. The survival rate atthe end of the learning period is greater than 90%.

[0114] Nine days post-op, the hearts were removed from heparinized (500U) mice and euthanized with a lethal dose of pentobarbital (150 mg/kg).The hearts were analyzed by visual inspection of a cross-section of theheart (FIG. 2A), determination of heart to body weight ratio (FIG. 2B),and light microscopy of the cardiomyocytes in the left ventricular (FIG.2C), and activation of artiel natuiretic factor (ANF), a specificmolecular marker for hypertrophy (FIG. 3). Based on thesedeterminations, all trans-aortic constricted mice developed well-definedleft ventricular hypertrophy.

[0115] To determine whether tyrphostin AG490 could reverse thehypertrophy induced by traverse aortic constriction, tyrphostin AG490 (5μM) was administered to the mice, intra-peritoneal, 24 hours beforebeing subjected to transverse aortic constriction and every 24 hoursthereafter for the duration of the study (9 days). Chronicadministration of tyrophostin AG490 caused a remarkable reversal ofhypertrophy (see FIGS. 2A, 2B, 2C, and 3).

EXAMPLE 2

[0116] This example demonstrates that administration of Jak2 affordedcardioprotection against ischemia-induced changes in myocardialperformance by inhibition of Jak2.

[0117] Using spontaneously beating working hearts that were not paced,the absolute values and the first derivative of developed pressure wereprogressively decreased with reperfusion, as expected (FIG. 4A). Theinhibitor, tyrphostin AG490, at both 5 and 50 μmol/L, was able toprovide cardioprotection to approximately the same degree. This wasparticularly true during the first 60 min. of reperfusion, when thedP/dt value was not lowered, and developed pressure was minimallylowered, in the treated groups. The baseline value for dP/dt increasedslightly in high (50 μmol/L) concentrations of tyrphostin AG490. Inaddition, the slopes of the decay for the treated and untreated groupsafter 60 min. were similar. The values for both dP/dt and developedpressure in all treated groups were significantly higher than in theuntreated group. Developed pressure was notably higher in the tyrphostingroups subjected to 60 min. of reperfusion, R-60 (86±2.5 and 86±4.8compared with 64±3.2 mm Hg); 90 min. of reperfusion, R-90 (69±5 and72.7±5.7 compared with 46±3 mm HG); and 120 min. of reperfusion, R-120(60.85±4 and 53.75±7 compared with 38.66±2 mm Hg). dp/dt values weremarkedly higher in groups at both concentrations throughout most of thereperfusion period compared with the control reperfused group, thedifference being apparent at R-30 (3818±49.46 and 4156±238 versus3382±68.8), R-60 (3362±53.14 and 3840±140 versus 2878±237), R-90(2840±88 and 3194.7±228 versus 1842±162), and R-120 (2552±58.9 and2626±269 versus 1543±94).

[0118] To gain insight into the physiological basis for cardioprotectionafforded by tyrphostin AG490, the extent of cardiomyocyte infarct sizeand apoptosis were measured. On termination of treatment with tyrphostinAG490, hearts were immersed in 1% triphenyl tetrazolium solution inphosphate buffer (Na₂HPO₄ 88 mmol/l, NaH₂PO₄ 1.8 mmol/l) for 10 min. at37° C. and stored at −70° C. for processing. Frozen hearts (ventriculartissue) were sliced transversely in a plane perpendicular to theapicobasal axis into 0.5 mm thick sections, blotted dry, placed betweenmicroscope slides, and scanned on a Hewlett-Packard Scanjet 5 psingle-pass flatbed scanner. With the NIH 1.61 image processingsoftware, each digitized image was subjected to equivalent degrees ofbackground subtraction, brightness, and contrast enhancement forimproved clarity and distinctness. Risk (equivalent to total leftventricular muscle mass) and infarct zones of each slice were traced,and the respective areas were calculated in terms of pixels. The weightof each slice was then recorded to facilitate the expression of totaland infarct masses of each slice in grams. The risk and infarct volumesof each slice in cubic centimeters were then calculated on the basis ofslice weight to correct for any errors due to nonuniformity of heartslice thickness. The risk volumes and infarct volumes of each slice weresummed to obtain the risk and infarct volumes for the whole heart.Infarct size was taken to be the percent infarct volume/risk volume forany one heart.

[0119] Immunohistochemical detection of apoptotic cells was carried outby use of terminal dUTP nick end-labeling (TUNEL), in which residues ofdigoxigenin-labeled dUTP are catalytically incorporated into the DNA byterminal deoxynucleotidyl transferase II. The cells were incubated witha sheep polyclonal anti-digoxigenin antibody followed by aFITC-conjugated rabbit anti-sheep IgG as a secondary antibody. The heartsections were washed in PBS 3 times, blocked with normal rabbit serum,and incubated with mouse monoclonal antibody recognizing cardiac myosinheavy chain (Biogenesis Ltd) followed by staining with TRIRC-conjugatedrabbit anti-mouse IgG (200:1 dilution, Dalco, Japan). The fluorescencestaining was viewed with a confocal laser microscope (Olympus Co). Theapoptoic cells were counted and expressed as percentage of total myocytepopulation.

[0120] Administration of tyrphostin AG490 reduced myocardial infarctsize (FIG. 4B) and caused a marked lowering of apoptotic cell death(FIG. 4C), thereby, attributing, at least in part, to the recovery ofcontractile function upon treatment with tyrphostin AG490.

EXAMPLE 3

[0121] This example demonstrates upregulation of rat heartangiotensinogen mRNA during ischemia/reperfusion.

[0122] Ischemia was induced by a modified Langendorf-reperfusion methodin rat hearts. Hearts from adult male rats were randomly divided into 4groups and subjected to ischemia/reperfusion. In the ischemic group,hearts were perfused with Krebs-Henseleit buffer for 60 minutes,followed by 30 minutes of global ischemia. In the ischemic/reperfusedgroup, hearts were perfused for 60 minutes, followed by 30 minutes ofglobal ischemia and 120 minutes of reperfusion. Control group heartswere perfused for the same lengths of time.

[0123] Rat hearts subjected to ischemia/referfusion were tested todetermine whether activation of the renin-angiotensin system, asreflected by an increase in angiotensinogen mRNA, occurs in ischemicinjury. The level of angiotensinogen mRNA was analyzed by primerextension assay using gene-specific DNA probes. A DNA primer spanningthe complementary sequence of the rat angiotensinogen cDNA betweennucleotides 302 and 279 (5′-AGGAGATGAAAGGGGTGGATGTAT-3′) was end-labeledand used to evaluate the expression of angiotensinogen mRNA in total RNAisolated from the rat heart. The primer extension protocol was performedaccording to instructions provided by the supplier (Progema). Rat GAPDHcDNA specific primer was used as control. There was a marked increase inmRNA level after 30 minutes of ischemia and 120 minutes of reperfusion(FIG. 5A). The increase in mRNA was sensitive to blockage of the AT₁receptor, because pretreatment with losartan (L) reduced it almostentirely to the level of the control sample (C). The levels of theribosomal marker L32 mRNA, used as control, remained unchanged.

EXAMPLE 4

[0124] This example demonstrates STAT activation duringischemia/reperfusion.

[0125] Nuclear extracts were examined in hearts subjected to globalischemia to determine whether there is enhanced STAT binding activity tothe St domain of the angiotensinogen promoter. The nuclear heartextracts were examined by the electrophoretic gel mobility-shift assaythat exployed the use of the chemically synthesized oligonucleotidesequence of the St domain. The St-domain DNA probe for protein bindingwas a double-stranded oligonucleotide containing the sequence5′-GGGTtcCTGGAAGGG-3′ and complementary strand 5′-CCCTTCCAGgaACCC-3′,respectively. These probes were end-labeled by polynucleotide kinase and[γ-³²P]ATP. Binding reaction mixture containing 0.5 ng of labeled DNA(1,000 cpm), 2 μg of poly(dI-C), and 1-12 μg of protein in buffercontaining 20 mM Hepes, 3% glycerol, 1.5 mM MgCl₂, 1 mM DTT, 2 mM EDTAand 50 mM KCl, pH 7.5 was allowed to incubate at 4° C. for 30 min. Thereactions were analyzed by electrophoresis on 8% polyacrylanide gel in0.375× TBE (0.33 mM Tris borate, pH 8.7 and 1.0 mM EDTA). Afterelectrophoresis, the gels were dried and subjected to autoradiography.There was a strong St-domain/STAT binding activity in the heartssubjected to 30 min. ischemia/120 min. reperfusion, which was almostentirely abolished in losartan-treated heart, suggesting that losartan(L) treatment during perfusion resulted in loss of the activated STATparticipation in complex formation. (FIG. 5B). The activation of STATsand the consequent binding to the St-domain in the angiotensinogenpromoter accounts for the increase in transcription of angiotensinogenmRNA. Thus, the loss of STAT/DNA interaction and the reduction in theangiotensinogen mRNA levels (see FIG. 5A) due to losartan treatmentappear to be correlative.

[0126] To identify the STAT proteins that were activated in the ischemichearts, nuclear extracts were preincubated for 30 min with polyclonalantibodies against STAT1, STAT3, STAT5A, and STAT6 before adding theSt-domain DNA labeled probe. Examination of the reaction by gelmobility-shift assay showed that STAT5A and STAT6 DNA complexes wereprominently disrupted by antibodies against STAT5A and STAT6 (FIG. 5C).Therefore, STAT5A and STAT6 are activated in ischemic hearts.

EXAMPLE 5

[0127] This example demonstrates the effect of Jak2 inhibition onSTAT/DNA binding and angiotensinogen mRNA.

[0128] Rats were pretreated with 5 or 50 μmol/L of tyrphostin AG490 24 hprior to ischemia/reperfusion followed by chronic administration oftyrphostin AG490 during the process of ischemia/reperfusion. Aphosphotyrosine assay was performed. Briefly, nuclear extracts fromhearts subjected to ischemia/reperfusion in presence of absence oftyrphostin AG490 were immunoprecipitated with anti-phosphotyrosineantibodies (4G10). Fifty microliters of 50% protein A-agarose, prewashedin lysis buffer (Upstate Biotechnology) was then added and the mixturewas incubated for 2 hr at 4° C. Each sample was washed with washingbuffer containing 150 mM NaCl, 50 mM Tris-HCL (pH 7.4), 5 mM EDTA, 0.25%Triton X-100, 2 mM phenylmethylsulfonyl fluoride, aprotinin (0.2unit/ml), 1 mM Na₃VO₄, and 1 mM NaF. Samples were eluted in 2×Laernmli's sample buffer. Proteins were separated on a 7.5%SDS/polyacrylamide gel and transferred to nitrocellulose membrane,Nitropure (Micron Separations, Westboro, Mass.). Blots were probed withpolyclonal antibody against Jak2 and developed according to thechemiluminescence protocol. Administration of tyrphostin AG490 inperfusion medium was inhibitory at both 5 and 50 μmol/L forpliosphorylation of Jak2, which was activated readily in the ischemicheart in absence of the inhibitor (FIG. 6A).

[0129] When extracts from the same hearts were examined by gelmobility-shift assay for DNA binding, there was a total loss of STAT/DNAcomplex formation in the tyrphostin AG490 treated hearts (FIG. 6B).Treatment with tyrphostin AG490 also inhibited the stimulation of theangiotensinogen mRNA level that was observed in the ischemic tissues inabsence of the inhibitor (FIG. 6C). These results therefore stronglysuggest that activation of the Jak/STAT pathway, increases in theSTAT/angiotensinogen promoter binding activity, and the upregulation ofangiotensinogen mRNA all are casually related.

EXAMPLE 6

[0130] This example demonstrates Jak2 is a potent activator ofangiotensinogen gene expression.

[0131] Jak2 expression plasmid DNA was introduced via transfection intoliver cells in culture along with the angiotensinogen (ANG)/luciferasereporter DNA. Transfections were performed using FUGENE (BoehringerMannheim) to facilitate DNA uptake. A co-transfection assay withincreasing concentration of plasmid pTELJAK2 (ng) that expresses aconstitutively active Jak2 kinase and plasmid pANGLuc (1 μg) thatcarries the rat ANG promoter or plasmid pMANGLuc (1 μg) with asubstitution mutation in the conserved St-domain were delivered in humanhepatoma cell line HEPG2. After 48 hr, cells were collected and theluciferase activity of the reporter plasmids were evaluated usingstandard protocol (Luciferase assay TM-Promega).

[0132] Expression of angiotensinogen occurs in a concentration dependentmanner (FIG. 7).

EXAMPLE 7

[0133] This example demonstrates attenuation of angiotensinogen mRNAexpression in vivo by tyrphostin AG490.

[0134] Tyrphostin AG490 was administered to rats in vivo. A subcutaneouspump was surgically placed with a catheter extended intraperitoneally.Rats were daily treated with tyrphostin AG490 to achieve a 5 μMconcentration systemically. After 10 days, the animals were euthanizedusing approved animal protocols and the liver collected to isolate RNA.The isolated RNA was used to perform a northern blot and thenitrocellulose membrane was probed with the rat angiotensinogen cDNA.Loading control was performed using GAPDH as probe.

[0135] Administration of tyrphostin AG490 abolished the expression ofangiotensinogen gene product mRNA (FIG. 8).

EXAMPLE 8

[0136] This example demonstrates attenuation of hypertension bytyrphostin AG490.

[0137] Adult spontaneously hypertensive rats, which are geneticallypredisposed to hypertension, and normotensive rats were anesthetizedwith pentobarbital sodium (65 mg/kg ip) in preparation for surgery.Using aseptic rat surgical procedures, a groin incision was made toexpose inferior epigastric vessels along with femoral vessel. Teflonarterial catheter (ID 0.029 mm) was inserted into the left femoralartery. The catheter was tunneled under the skin and exited at the napeof the rat's neck and connected to a 26 G needle capped on a port andflushed with 1:3 heparin saline.

[0138] The rats were housed individually after surgery, and allowed 48hours to recover from the operation. Systolic and diastolic bloodpressure were directly measured using the connection to the port placedin the rat's neck to a manual transducer.

[0139] Administration of 5 micromoles of tyrphostin AG490 to thehypertensive rats was effective in lowering blood pressure to normallevels (FIG. 9).

1 3 1 24 DNA Artificial Primers 1 aggagatgaa aggggtggat gtat 24 2 15 DNAArtificial primers 2 gggttcctgg aaggg 15 3 15 DNA Artificial primers 3cccttccagg aaccc 15

We claim:
 1. A method for reducing hypertension in a mammal at risk for said hypertension comprising administering to said mammal an effective amount of a selective Jak2 inhibitor.
 2. A method as set forth in claim 1 wherein said Jak2 inhibitor is a tyrphostin.
 3. A method as set forth in claim 2 wherein said tyrphostin is Tyrphostin AG490.
 4. A method as set forth in claim 1 wherein said mammal is human.
 5. A method as set forth in claim 1 wherein said administration of the Jak2 inhibitor is systemic.
 6. A method as set forth in claim 1 wherein said composition is administered before damage from said hypertension occurs.
 7. A method as set forth in claim 1 wherein said effective amount produces a blood serum level between about 0.05 μM to about 10 μM
 8. A method as set forth in claim 7 wherein said effective amount produces a blood serum level preferably between about 1 μM to about 5 μM.
 9. A method for reducing heart failure in a mammal at risk for said heart failure comprising administering to said mammal an effective amount of a selective Jak2 inhibitor.
 10. A method as set forth in claim 9 wherein said Jak2 inhibitor is a tyrphostin.
 11. A method as set forth in claim 10 wherein said tyrphostin is Tyrphostin AG490.
 12. A method as set forth in claim 9 wherein said mammal is human.
 13. A method as set forth in claim 9 wherein said composition is administered before said heart failure occurs.
 14. A method as set forth in claim 9, wherein said composition is administered after said heart failure occurs to prevent further damage.
 15. A method as set forth in claim 9 wherein said administration of the Jak2 inhibitor is systemic.
 16. A method as set forth in claim 9 wherein said effective amount is an amount that produces a blood serum level between about 0.05 μM to about 10 μM
 17. A method as set forth in claim 16 wherein said effective amount is an produces a blood serum level preferably between about 1 μM to about 5 μM. 